Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization

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1 FERTILITY AND STERILITY VOL. 74, NO. 6, DECEMBER 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization Donald E. Moore, M.D., Michael R. Soules, M.D., Nancy A. Klein, M.D., Victor Y. Fujimoto, M.D., Kathy J. Agnew, B.S., and David A. Eschenbach, M.D. Department of Obstetrics and Gynecology, Division of Gynecology, and Division of Reproductive Endocrinology and Infertility, University of Washington School of Medicine, Seattle, Washington Objective: To assess the impact of individual bacteria isolated from the vagina and tip of the embryo transfer catheter on live-birth rates. Design: Prospective clinical study. Setting: Infertility outpatient clinic of a university hospital. Patient(s): Ninety-one women undergoing IVF-ET. Intervention(s): Cultures were obtained from the vagina for aerobic and anaerobic bacteria at the time of both sonographic egg retrieval and embryo transfer and from the tip of the embryo transfer catheter. Doxycycline treatment was started after egg retrieval. Main Outcome Measure(s): The live birth of one or more neonates. Result(s): Doxycycline had no substantial impact on the recovery of individual vaginal bacteria or on bacterial vaginosis. An increase in live-birth rate was associated with the recovery of hydrogen peroxide producing Lactobacillus from the vagina (P 0.01) and from the embryo transfer catheter (P 0.01). In contrast, a reduction in live-birth rate was associated with recovery of Streptococcus viridans (S. viridans) from the embryo transfer catheter tip (P 0.04). Conclusion(s): In the setting of IVF-ET, prophylactic doxycycline had little effect on vaginal bacteria. Specific bacteria recovered from the embryo transfer catheter appear associated with a detrimental or beneficial effect or with no effect on live-birth rates. (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) Key Words: Infection, IVF-ET, prophylactic antibiotics, transfer catheter Received February 14, 2000; revised and accepted July 3, Presented as a poster at the American Society for Reproductive Medicine s 55th Annual Meeting in Toronto, Canada, September 19, Reprint requests: Donald E. Moore, M.D., Division of Reproductive Endocrinology and Infertility, 4225 Roosevelt Way N.E., Suite 305, Seattle, Washington (FAX: ; sunbeam@u.washington.edu) /00/$20.00 PII S (00) Clinical pelvic infection is infrequent after IVF-ET procedures (1 3). However, because IVF-ET procedures involve needle puncture of the vagina and catheter placement of embryos through the cervix, contamination is possible from vaginal-cervical microorganisms. Vaginal antiseptics usually are not used during egg retrieval or embryo transfer to avoid injury to the eggs or embryos. Instead, most in vitro fertilization (IVF) programs use systemic antimicrobials to prevent clinical pelvic infection. Infection of the oocyte or developing embryo also could occur during IVF procedures. A subclinical infection could result from potential pathogens in semen. Most ejaculated semen are colonized with microorganisms (4) introduced into the gamete culture media when semen is added. However, gentle swim-up methods used to separate sperm from seminal fluid (5) and antimicrobials added to media inhibit the growth of these bacteria. We propose that minimal inflammation in response to microorganisms that enter the endometrium from the cervix during embryo transfer provides another mechanism that could damage the developing embryo and prevent pregnancy. Antimicrobials in the culture media probably provide little inhibition to the potentially large number of bacteria that could contaminate the embryo transfer catheter when it traverses the cervix. In fact, a 50% reduction in pregnancy rate has been reported in subjects with bacteria compared with those without bacteria isolated from the embryo transfer catheter tip (6 8). 1118

2 In most women, Lactobacillus species is the predominant bacteria in the vagina. Lactobacillus appears to reduce the number of other bacteria in the vagina by maintaining a low ph and by producing hydrogen peroxide (H 2 O 2 ). H 2 O 2 - producing Lactobacillus inhibits the growth of bacteria (9) and virus (10) in vitro and perhaps in vivo (11). Thus, women with vaginal flora dominated by low-virulence Lactobacillus, particularly strains that produce H 2 O 2, would be expected to have few virulent bacteria. In contrast, women with bacterial vaginosis have few H 2 O 2 -producing Lactobacillus and an increased concentration of other bacteria (12). In fact, an increased risk of pregnancy loss before 6 weeks was reported in women with bacterial vaginosis (BV) undergoing IVF (13). We conducted a prospective observational study to examine the impact of doxycycline on vaginal microbes, the association between the isolation of microorganisms from the vagina and the tip of the transfer catheter, and the association between specific microbes and the livebirth rate. MATERIALS AND METHODS Patient Population The study population consisted of patients undergoing IVF-ET cycles between May 1997 and May 1998 at the Fertility and Endocrine Center at the University of Washington. The established IVF-ET protocols remained constant during the study period. Only one cycle per patient was used in the analyses, although it was not necessarily the subject s first IVF cycle. Only subjects who underwent transvaginal sonographic egg retrieval (SER) under conscious sedation and had an embryo transfer were included in the study. Subjects signed a consent form approved by the University of Washington Human Subjects Review Committee before study entry. Subjects ranged in age from years. Donors or recipients of donor eggs were specifically excluded. Each woman underwent one of three standard ovulation induction protocols. In the luteal leuprolide acetate protocol, each woman began a daily injection of leuprolide acetate in the midluteal phase that continued until hcg was given. Ten or more days after the initiation of leuprolide acetate and the suppression of the ovaries, gonadotropins (FSH or FSH and LH) were initiated and continued until the day human chorionic gonadotropin (hcg) was given. In the microdose leuprolide acetate protocol, low dose leuprolide acetate and gonadotropins were started 2 days after completion of 21 days of daily oral contraceptives. Both were continued until the day hcg was administered. In the straight gonadotropin protocol, no leuprolide acetate was used, and gonadotropins were started on day 3 of the menstrual cycle and continued until the day hcg was administered. In each of the protocols, once two lead follicles were mm in mean diameter, hcg (10,000 IU) was given FIGURE 1 Timeline for 91 women undergoing IVF procedures, study procedures for isolation of bacteria, and the timing of prophylactic oral doxycycline. SER sonographic egg recovery; ET embryo transfer. that evening, and SER was performed by one of four physicians 36 hours later. At SER, swabs were obtained from the vagina for culture. The vagina then was washed extensively with normal saline, and the SER was performed. Doxycycline (100 mg b.i.d.) was given for 5 days, starting the evening of egg retrieval or, if the bladder was not catheterized, the morning after egg retrieval (Fig. 1). Embryo transfer (ET) occurred 2 days after the SER if the woman was 38 years of age, and 3 days after the SER if the woman was 38 years of age. In the latter age group, assisted hatching (the process of developing a small opening in the zona by acidified Tyrode s solution just minutes before embryo transfer) was performed, and methylprednisolone 16 mg a day was started the day of the egg retrieval and continued for 4 days. The gamete and embryo culture media was human tubal fluid (HTF) medium plus 10% synthetic serum substitute with penicillin and streptomycin added (Irvine Scientific, Santa Ana, CA). Swabs for culture again were obtained from the vagina at embryo transfer. The vagina and cervix then were washed with gamete culture media. Cervical mucus was aspirated from the cervical canal. During the study period, two different transfer catheters were used, at the discretion of the physician. The most common was a Tefcat catheter (Cook ObGyn Co., Spencer, IN), which is relatively stiff. The other was a softer Cook coaxial catheter (Cook ObGyn Co.). Daily progesterone was started on the fourth day after the hcg, either as an i.m. injection or as a vaginal preparation (Crinone 8% vaginal gel, Wyeth-Ayerst Laboratories, Philadelphia, PA). Culture Collection and Microbial Isolation After insertion of a vaginal speculum, three sterile cotton swabs were rubbed on the vaginal wall. One swab was rolled FERTILITY & STERILITY 1119

3 TABLE 1 Comparison of selected demographic and reproductive characteristics between women with and without S. viridans and hydrogen peroxide producing Lactobacillus. S. viridans H 2 O 2 -producing Lactobacilli Parameter Bacteria present Vagina Catheter tip Vagina Catheter tip Age of women Yes No a Months of infertility Yes No No. (%) of couples previously pregnant together Yes 19/50 (38%) 6/7 (35%) 5/27 (19%) 1/10 (10%) No 9/39 (23%) 22/72 (31%) 23/62 (37%) 27/79 (34%) Note: Values are means SEM unless otherwise stated. a P 0.02, comparing age among subjects with and without H 2 O 2 -producing Lactobacillus isolated from the ET catheter tip. on a glass slide for Gram staining, and the flora later was scored as normal, as intermediate, or as BV based on the concentrations of Lactobacillus, Gardnerella or bacteroides, and Mobiluncus morphotypes as previously described by Nugent (14). Two swabs were placed deep into anaerobic transport media (Port-A-Cul, Becton Dickenson, Cockeysville, MD). After completion of the embryo transfer, the catheter tip was cut with sterile scissors and placed tip first into a second anaerobic transport media. The two vaginal swabs, each holding approximately g of vaginal fluid (12), were placed in 1.5 ml of phosphate-buffered saline (PBS) solution and vortexed vigorously. Serial 1:10 dilutions were carried out, and the resulting dilutions were inoculated onto agar suitable for the recovery of aerobic and anaerobic microorganisms, yeast, and genital mycoplasms as previously reported (12). Culture plates for aerobic (facultative), yeast, and genital mycoplasma microorganisms were incubated at 37 C in 5% 10% CO 2 and examined after hours. Culture plates for anaerobic microorganisms were incubated within an anaerobic glove box at 37 C for 5 uninterrupted days. Microorganisms were identified as previously reported (12). Hydrogen peroxide production by Lactobacillus was tested as previously described (15). Definition Chemical pregnancy was defined as a positive hcg titer, and clinical pregnancy, as cardiac motion detected by ultrasound. A live birth (LB) was defined as the live birth of one or more neonates. Statistical Analysis The 2 test of significance was used to test the relationships between specific bacteria and live-birth rates and to determine the likelihood that these events occurred by chance. The Fisher exact probability test was used when the total sample size was less than 20. The Mann-Whitney U-test and stratified analyses were used to test for factors that might confound these relationships (Table 1). A P value of 0.05 was considered statistically significant. All values are presented as means standard error unless otherwise stated. RESULTS At least one culture was obtained from each of 127 individuals: 113 had vaginal cultures at SER, 95 had vaginal cultures at ET, 106 had cultures of the ET catheter tip, and 91 had a set of all three cultures. Only the results from the latter 91 are presented. The mean age of the 91 individuals was years, with a range of years. Primary infertility was present in 46 women. The primary infertility diagnosis was male factor in 40%, tubal factor in 30%, endometriosis in 20%, unexplained infertility in 2%, amenorrhea in 2%, and other factors in 6%. The prevalence and concentration of microorganisms recovered at SER before doxycycline are compared in Table 2 with those recovered at ET, hours after doxycycline was started. The comparison reflects the impact of doxycycline on individual vaginal microorganisms. The proportion of women with individual vaginal microorganisms recovered at SER and at ET was similar for all microorganisms (Table 2). The log concentration in 1 ml of vaginal fluid of individual vaginal microorganisms in Table 2 also was similar between SER and ET. The most common microorganisms isolated from the vagina at both SER and ET were Lactobacillus species, Streptococcus viridans, Enterococcus, and Staphylococcus epidermis, followed by Ureaplasma urealyticum, Escherichia coli, anaerobic gram-positive cocci, Prevotella spp., and Mycoplasma hominis. Bacterial vaginosis and intermediate flora based on Gram stain criteria were present in a similar number of women at SER and ET. H 2 O 2 -producing Lactobacillus was recovered from the vagina in 5 (12%) of the 41 women with BV or intermediate 1120 Moore et al. Bacteria and IVF outcome Vol. 74, No. 6, December 2000

4 TABLE 2 The prevalence and concentration of microbial isolates from the vagina of 91 women before (sonographic egg retrieval) and after (embryo transfer) doxycycline therapy. Sonographic egg retrieval (before doxycycline) Embryo transfer (after doxycycline) Microorganism No. (%) Mean log concentration a No. (%) Mean log concentration a H 2 O 2 -producing Lactobacillus 27 (30%) (31%) 7.1 H 2 O 2 -nonproducing Lactobacillus 18 (20%) (15%) 7.4 S. viridans 46 (51%) (56%) 6.7 Enterococcus 54 (59%) (60%) 7.0 Staphylococcus epidermidis 49 (54%) (51%) 3.7 Escherichia coli 31 (34%) (26%) 7.3 Group B streptococci 10 (11%) (13%) 6.6 Anaerobic gram cocci 29 (32%) (27%) 5.9 Prevotella species 26 (29%) (21%) 4.8 Ureaplasma urealyticum 32 (35%) (34%) 9.1 Mycoplasma hominis 16 (18%) (25%) 11.0 Intermediate flora 23 (25%) 31 (34%) BV 12 (13%) 10 (11%) Note: Not included in this table are the following microorganisms recovered from fewer than 10 women: other Lactobacillus species (which failed to grow in H 2 O 2 media), Candida albicans and other yeast, black-pigmented anaerobic gram-negative rods, Gardnerella vaginalis, Staphylococcus aureus, other gram-negative rods, Bacteroides fragilis, and Bacteroides ureolyticus. The P values that compared the number of women with microorganisms and the concentration of microorganisms recovered at sonographic egg recovery with those at embryo transfer were all nonsignificant (P 0.05). a Mean log concentration of microorganisms per milliliter of vaginal fluid. flora and in 22 (46%) of the 50 women with normal flora by Gram stain (P 0.001). Prevotella species was recovered from the vagina of 14 (34%) of the women with BV or intermediate flora and of 5 (10%) of the women with normal flora according to Gram stain (P 0.01). The mean log concentration of Prevotella in the vagina was higher (log ) in those with BV or intermediate flora than in those with normal flora according to Gram stain (log , P 0.05). S. viridans and other microbes in the vagina were not related to BV or intermediate Gram stain flora. The isolation of the microorganisms listed in Table 2 from the vagina was not significantly related to the isolation of H 2 O 2 -producing Lactobacillus from the vagina. For example, S. viridans was recovered from 41% of 27 women with H 2 O 2 -producing Lactobacillus and from 55% of 64 women without H 2 O 2 -producing Lactobacillus in the vagina at SER (P 0.3). In contrast, a correlation did occur between the concentration of S. viridans or H 2 O 2 -producing Lactobacillus in the vagina and the recovery of these bacteria from the embryo transfer catheter tip. S. viridans was recovered from the catheter tip of 15 of 51 patients with S. viridans in the vagina. The log concentration of the S. viridans in the vagina of these 15 subjects was higher ( ) than that of the other 36 patients without S. viridans on the transfer catheter tip ( , P 0.002). The log concentration of H 2 O 2 - producing Lactobacillus in the vagina was for the 9 patients with and for the 19 patients without H 2 O 2 -producing Lactobacillus recovered from the catheter tip (P 0.05). The association between individual microorganisms isolated from the vagina or embryo transfer catheter tip and the live-birth rate is presented in Table 3. All women had at least one microorganism isolated from the vagina and 44 (48%) had at least one microorganism isolated from the embryo transfer catheter tip. The live-birth rate was significantly higher when H 2 O 2 -producing Lactobacillus was recovered from the vagina at ET (50%) than when it was not (21%; P 0.01). The live-birth rate was also associated with the recovery of H 2 O 2 -producing Lactobacillus from the embryo transfer catheter tip (P 0.01). The live-birth rate was slightly lower when S. viridans was recovered (24%) than when it was not recovered (38%) from the vagina at ET (P 0.2). However, the live-birth rate was markedly lower when S. viridans was recovered from the embryo transfer catheter tip (6%) than when it was not (35%, P 0.04). The recovery of other individual microorganisms from the vagina or the embryo transfer catheter tip or an intermediate or BV Gram stain score was not associated with the live-birth rate. Of interest, the live-birth rate was also associated with the recovery of H 2 O 2 -producing Lactobacillus from the vagina (48%) vs. no H 2 O 2 -producing Lactobacillus (22%) at SER (P 0.02). A live birth occurred for 7 (37%) of the 19 women with any microorganisms in the transfer catheter (excluding FERTILITY & STERILITY 1121

5 TABLE 3 Comparison of live-birth rate with the recovery of selected microorganisms from the vagina or embryo transfer catheter tip during the sonographic egg recovery and embryo transfer. Live-birth rate associated with recovery of the microorganism Microorganism in the vagina at embryo transfer Microorganism on the transfer catheter tip Microorganism Isolated Not isolated P value Isolated Not isolated P value H 2 O 2 -producing Lactobacillus 14/28 (50%) 13/63 (21%) /10 (70%) 20/81 (25%) 0.01 H 2 O 2 -nonproducing Lactobacillus 1/14 (7%) 26/77 (34%) /8 (38%) 24/83 (29%) 0.9 S. viridans 12/51 (24%) 15/40 (38%) 0.2 1/17 (6%) 26/74 (35%) 0.04 Enterococcus 14/55 (25%) 13/36 (36%) 0.4 3/11 (27%) 24/80 (30%) 1.0 Staphylococcus epidermidis 18/46 (39%) 9/45 (20%) /4 (75%) 24/87 (28%) 0.1 Other facultative isolates 13/46 (28%) 14/45 (31%) 0.9 2/6 (33%) 25/85 (29%) 1.0 Anaerobic gram isolates 8/25 (32%) 19/66 (29%) 0.9 5/11 (45%) 22/80 (28%) 0.4 U. urealyticum and M. hominis 7/31 (23%) 20/60 (33%) 0.4 0/3 (0%) 27/88 (31%) 0.6 Intermediate flora 7/31 (23%) 18/50 (36%) 0.3 BV 2/10 (20%) 18/50 (36%) 0.6 H 2 O 2 -producing Lactobacillus and S. viridans) and in 12 (26%) of the 47 women with no microorganisms isolated from the ET tip (P 0.6; Table 4). However, a bimodal distribution was present when we examined the isolation of individual bacteria from the ET tip. The live-birth rate was greater when H 2 O 2 -producing Lactobacillus (without S. viridans) was isolated from the ET catheter tip (88%) than when no bacteria were isolated from the catheter tip (26%, P 0.003). Conversely, the live-birth rate was less when S. viridans (and no H 2 O 2 -producing Lactobacillus) was isolated (7%) than when no bacteria were isolated from the ET catheter tip (26%, P 0.2). The livebirth rate for those with H 2 O 2 -producing Lactobacillus (88%) compared with those with S. viridans isolated from the ET catheter tip (7%) were highly different from each other (P ). No live births occurred in the two individuals with both S. viridans and H 2 O 2 -producing Lactobacillus isolated from the transfer catheter. We also analyzed the early pregnancy loss by a comparison of chemical and clinical pregnancy with the isolation of bacteria from the ET catheter tip. Pregnancy continued with a live birth for all 7 patients with a chemical pregnancy and H 2 O 2 -producing Lactobacillus on the transfer catheter tip. Of the 31 patients with a chemical pregnancy and no H 2 O 2 - producing Lactobacillus or S. viridans on the ET catheter tip, 22 had a clinical pregnancy, and 19 had a live birth. Of the 3 patients with a chemical pregnancy with S. viridans but no H 2 O 2 -producing Lactobacillus isolated from the ET catheter tip, 2 had a clinical pregnancy, and 1 had a live birth. The early pregnancy loss associated with H 2 O 2 -producing Lactobacillus or other microorganism on the catheter tip was not significant in these small groups. In Table 1, we present three variables that might confound the findings of the live-birth rate or the recovery of H 2 O 2 -producing Lactobacillus or S. viridans from the transfer catheter tip. Only age had an effect on the pregnancy rates in this study. Pregnancy occurred in 11% of women 38 years of age and in 38% of women 38 years of age (P 0.02). A stratified analysis of the women 38 years of age (excluding the two women who also had S. viridans in the transfer catheter) was performed. The live-birth rate of women 38 years of age was 88% in the 8 women with and TABLE 4 Live-birth rates in groups stratified by the isolation of selected bacteria from the tip of the embryo transfer catheter. Transfer catheter tip Bacteria isolated from the tip of the embryo Live birth, n (%) No live birth Totals H 2 O 2 -producing Lactobacillus 7 (88%) 1 8 (excluding S. viridans) Any microorganism (excluding H 2 O 2-7 (37%) producing Lactobacillus and S. viridans) None 12 (26%) a S. viridans isolated (excluding H 2 O 2-1 (7%) b producing Lactobacillus) Both S. viridans and H 2 O 2 -producing 0 (0%) 2 2 Lactobacillus Totals 27 (30%) a P 0.003, b P , compared with H 2 O 2 -producing Lactobacillus isolated (excluding S. viridans) Moore et al. Bacteria and IVF outcome Vol. 74, No. 6, December 2000

6 39% in the 46 women without H 2 O 2 -producing Lactobacillus isolated from the ET catheter tip (P 0.03). Thus, after removal of the confounding variable of age 38 years, the positive effect of H 2 O 2 -producing Lactobacillus on livebirth rate remained. Months of infertility and a previous pregnancy in the couple (Table 1) and the type of catheter tip and cause of the infertility (data not shown) were not associated with live birth, H 2 O 2 -producing Lactobacillus, or S. viridans on the catheter tip. DISCUSSION The overall 30% live-birth rate for the total population of 91 women undergoing IVF-ET is comparable to that of other reports (16). Women 38 years of age had a higher live birth rate than older women, also similar to other reports (16). Lactobacillus is a relatively nonvirulent bacteria. In many women, Lactobacillus species comprise 90% 95% of the total bacterial count in the vagina (12). Lactobacillus probably inhibits the growth of other potentially virulent bacteria in the vagina by producing lactic acid and hydrogen peroxide (H 2 O 2 ). Lactic acid helps maintain the vaginal ph at 4.5, which is inhibitory to the growth of most microbes. H 2 O 2 inhibits microbes with low levels of H 2 O 2 -scavenging enzymes, such as catalase. Further, H 2 O 2 combined with halide ions and peroxidase (both are present in the vagina) represents a potent system of in vitro and in vivo killing of bacteria (9, 11, 12) and virus (10, 17). In our study, H 2 O 2 -producing Lactobacillus in the vagina appeared to positively impact the live-birth rate among women undergoing IVF. Women with H 2 O 2 -producing Lactobacillus in the vagina were the most likely to have H 2 O 2 - producing Lactobacillus on the ET catheter tip. Women with an H 2 O 2 -producing Lactobacillus dominant vaginal flora would be expected to have only a few potential pathogenic organisms, in low concentration, in the vagina and cervix. Under these conditions, the likelihood of transfer of virulent organisms into the endometrium during embryo transfer would be low. Conversely, women without H 2 O 2 -producing Lactobacillus in the vagina would be expected to have an increased number of potentially pathogenic bacteria in the vagina (12) and cervix that could be transferred into the endometrium. Once in the endometrial cavity, virulent bacteria could stimulate macrophages and other immune cells and induce a proinflammatory cytotoxic response. Even a small inflammatory response in the endometrium would be expected to attract neutrophils and injure the embryo or prevent implantation. A negative effect occurred on live-birth rate when S. viridans was recovered from the catheter tip. The lowest live-birth rate (6%) occurred in the 17 women with S. viridans recovered from the catheter tip. Some species of S. viridans are virulent and cause heart valve damage, endocarditis, and intra-abdominal abscess. In previous studies, a 50% reduction in pregnancy rate occurred when bacteria were recovered from the tip of the embryo transfer catheter (6 8, 12). However, these studies did not report a positive effect on pregnancy of bacteria on the catheter tip. It is of interest that the dominant microorganisms differed among the studies. The dominant microorganisms were group D streptococcus and E. coli in Kuwait (6), E. coli in France (7), and S. viridans, Enterococcus, S. epidermidis, and H 2 O 2 -producing Lactobacillus in the United States. Of interest, recovery of no bacteria from the ET catheter tip resulted in a pregnancy rate of 26%, whereas recovery of bacteria other than H 2 O 2 -producing Lactobacillus or S. viridans resulted in a live-birth rate of 47%. This difference is not statistically significant (P 0.2) and may not represent a true difference. However, an examination is needed of whether difficult to culture or a low concentration of bacteria were actually present through use of polymerase chain reaction (PCR) to identify ribosomal DNA conserved in all bacteria (18). Doxycycline as used in this study had no effect on vaginal flora (Table 2). Doxycycline and other antimicrobials are commonly used to prevent clinical pelvic infection following invasive procedures, such as SER and ET. However, doxycycline has not been rigorously studied to determine its potential impact on vaginal flora or its impact on IVF pregnancy rates. The time between the start of doxycycline and embryo transfer was only 24 to 60 hours, and a longer time may be needed for antibiotics to impact vaginal flora. However, it was reassuring that doxycycline did not detrimentally affect vaginal flora. Further study is needed of antibiotics to determine whether they actually reduce virulent bacteria, such as S. viridans, and more important, to confirm that they do not reduce protective bacteria, such as Lactobacillus. Recently, it was reported that ceftriaxone and metronidazole given intravenously at egg retrieval in IVF-ET cycles may reduce bacteria on the transfer catheter and improve the pregnancy rate (8). However, a control group was not used in this study, H 2 O 2 -producing Lactobacillus was not reported, and the overall pregnancy rate was lower than in a previous study by the same group (P 0.2) (6). A strategy different than prophylactic antibiotics would be to induce the growth of H 2 O 2 -producing Lactobacillus in the vagina before embryo transfer. At present, this latter possibility has not been accomplished on any practical basis but is under study. There are several weaknesses in our study. The small numbers may have produced a beta error. A larger study would allow for more certainty of the conclusions. The ET tip culture recovery rate could be increased by the addition of liquid culture media instead of the solid media used in this report or by using PCR to detect bacterial DNA. FERTILITY & STERILITY 1123

7 Additionally, it was unusual to find a population of young women in which the prevalence of Lactobacillus in the vagina was only 50% and the prevalence of H 2 O 2 -producing Lactobacillus was only 30%. In most populations without genital infection, the prevalence of Lactobacillus is 70% 80%, and the prevalence of H 2 O 2 -producing Lactobacillus is 50% 60% (12, 15). The low rate of H 2 O 2 -producing Lactobacillus recovery in our population could have resulted from prior and/or repetitive antimicrobial therapy. H 2 O 2 -producing Lactobacillus would be infrequent in patients with BV or even intermediate flora as shown in this and other studies (12). Patients with BV usually have few H 2 O 2 -producing Lactobacillus and an increased concentration of facultative and anaerobic bacteria (12, 15). Bacterial vaginosis was present in 11% of this population, a rate lower than the 23% rate of BV often noted in pregnancy (12). An additional 34% of our study patients had intermediate flora. Thus, 44% of this population would be expected to have no or a low concentration of H 2 O 2 -producing Lactobacillus in the vagina. Bacterial vaginosis may lead to a higher preclinical pregnancy loss (before 6 weeks of gestation) as seen in a recent study (13). The data in that study suggested that BV could affect the pregnancy between implantation and 6 weeks of gestation (13), an effect not discerned in our smaller study. Finally, evidence of inflammation following embryo transfer needs to be documented to further examine our hypothesis that inflammation is the mechanism by which bacteria may effect the pregnancy. Interleukin-8 (IL-8), a potent neutrophil attractor, and interleukin-6 (IL-6) in the vagina have been related to amniotic fluid infection and preterm delivery during pregnancy (19, 20). The association of these proinflammatory cytokines after ET and a failure in live birth would strengthen this hypothesis. The impact of bacteria on live-birth rate is of considerable interest. If virulent bacteria could be identified in the vagina that affect live-birth rate, treatment of S. viridans alone could possibly increase the live-birth rate in our population from 30% to 38%. More important the induction of H 2 O 2 -producing Lactobacillus into the vagina could perhaps have an even greater impact on live-birth rate because 14 of the 27 live births occurred in the 30% of the women with H 2 O 2 -producing Lactobacillus in the vagina. Acknowledgments: The authors Jennifer Withrow, M.S., research coordinator in the Department of Obstetrics and Gynecology, for day-to-day operations, management of the patients and specimens, and data collection; and Soe Soe Thwin, M.S., research data manager in the Department of Obstetrics and Gynecology, for data entry. References 1. Sauer MV, Paulson RJ. Pelvic abscess complicating transcervical embryo transfer. Am J Obstet Gynecol 1992;166: Ashkenazi J, Farhi J, Dicker D, Feldberg D, Shalev J, Ben-Rafael Z. Acute pelvic inflammatory disease after oocyte retrieval: adverse effects on the results of implantation. Fertil Steril 1994;61: Yaron Y, Peyser M, Samuel D, Amit A, Lessing J. Infected endometriotic cysts secondary to oocyte aspiration for in vitro fertilization. Hum Reprod 1994;9: Hillier SL, Rabe LK, Muller CH, Zarutskie PW, Kuzan FB, Stenchever MA. Relationship of bacteriologic characteristics to semen indices in men attending an infertility clinic. Obstet Gynecol 1990;75: Kuzan FB, Hillier SL, Zarutskie PW. Comparison of three wash techniques for the removal of microorganisms from semen. Obstet Gynecol 1987;70: Egbase PE, Al-Sharhan M, Al-Othman S, Al-Mutawa M, Udo EE, Grudzinskas JG. Evidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical pregnancy rate following in-vitro fertilization and embryo transfer. 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Bacteria and IVF outcome Vol. 74, No. 6, December 2000